1. Technical Field
This invention relates to human cytomegalovirus (CMV), and in particular to methods and compounds for modifying immune responses to CMV in mammals. Specifically, the invention relates to single, tandem multiple and modified CMV proteins which can be expressed in cells using a vector such as an MVA vector or otherwise administered and to methods and compounds for expressing and using these proteins. The compounds of this invention are capable of augmenting immunity to CMV, for example by directing human cytotoxic T lymphocytes (CTL) to recognize and lyse human cells that are infected with CMV. Therefore, vaccines formulated using those compounds also are provided by this invention.
2. Description of the Background Art
The CMV genome is relatively large (about 235 k base pairs) and has the capacity to encode more than two hundred proteins. CMV consists of a nuclear complex of double-stranded DNA surrounded by capsid proteins having structural or enzymatic functions, and an external glycopeptide- and glycolipid-containing membrane envelope. CMV is a member of the herpes virus family and has been associated with a number of clinical syndromes. The major human cellular immune response targets of CMV have been described: pp65, pp150 and IE1. See, for example, U.S. Pat. Nos. 6,133,433 and 6,242,567. Glycoprotein B (gB) also is a CMV target recognized by the humoral immune system.
CMV infection is relatively common and is usually self-limiting in the healthy, immunocompetent child or adult (L. Rasmussen, Curr. Top. Microbiol. Immunol. 154:221–254, 1990), however the viral genome is not cleared from the individual and remains in a latent state. CMV persists in this latent state for life, under control and surveillance of cell-mediated immunity against CMV. In addition, approximately 10% of all newborn infants carry CMV. The virus can cause severe congenital disease in the fetus or infant. Some of these newborn infants suffer congenital birth defects. Others carry cytomegalovirus for some time after birth before they show symptoms of disease. CMV is a common cause of mental retardation in children who acquire the infection in utero from mothers carrying an active infection.
Several studies have begun to question whether persistent and apparently asymptomatic CMV infection in an otherwise healthy adult poses health risks in certain individuals. For example, individuals who have undergone coronary angioplasty sometimes subsequently develop restenosis as a result of arterial remodeling. In one study, about one third of such patients with restenosis had detectable CMV DNA in their arterial lesions (E. Speir et al., Science 265:391–394 (1994)). In another study, CMV seropositive patients were five times more likely to develop restenosis than their seronegative counterparts (Zhou et al., New England J. Med. 335:624–630 (1996)). These studies suggest that decreasing the number of CMV-infected host cells can benefit certain individuals with latent CMV infection, as well as those with an active infection.
CMV is an important consideration in the treatment of patients suffering from Acquired Immunodeficiency Syndrome (AIDS). The defining complication is viral retinitis, which, if left untreated, can lead to blindness. Historically, CMV disease has been one of the more devastating of the opportunistic infections that beset HIV-1-infected individuals. Disease manifestations of CMV viremia which appear as the CD4+ T cell counts drops below 100/mm3 include encephalitis, enteritis and pneumonia. At autopsy, there is multi-organ involvement of CMV disease in a great many AIDS patients who suffered from severe CMV retinitis. Patients infected with CMV often suffer impairment of at least some of their vital organs, including the salivary glands, brain, kidney, liver and lungs. Furthermore, CMV is associated with a wide spectrum of classical syndromes including mononucleosis and interstitial pneumonia.
CMV also has an oncogenic potential and a possible association with certain types of malignancies, including Kaposi's sarcoma. Recent studies have shown that CMV antigens are found in association with glioma cells and other brain tumors and with colorectal cancer. Harkins et al., Lancet 360(9345): 1557–1563, 2002; Cobbs et al., Cancer Res. 62(12): 3347–3350, 2002. CMV therefore may be responsible for part of the malignant transformation process or have some role in the progression of disease.
Since the first use of hematopoietic stem cell transplant as a therapy for hematological malignancies, one of the main infectious complications during the first one hundred days of recovery is pneumonia caused by CMV infection. CMV causes serious opportunistic infection in immunocompromised patients including but not limited to hematopoietic stem cell transplant patients, bone marrow transplant patients, solid organ transplant patients, HIV patients, gestational fetuses, infants and the like. Methods for vaccinating these patients in particular, and certain other individuals such as coronary angioplasty patients and women of child-bearing years, prophylatically or subsequent to infection with CMV to augment the CMV-specific cellular immune response are needed in the art. Strategies both to prevent primary infection and to aid in controlling viremia in patients already carrying CMV are needed, particularly in immunosuppressed or immunocompromised patients.
CMV can cause opportunistic infections which result in a variety of complications, for example immunosuppressed organ transplant patients. Prior to the use of antiviral chemotherapy, CMV infection had been responsible for a substantial proportion of post-bone marrow transplantation complications. Meyers et al., J. Infect Dis. 153:478–488, 1986. The use of drugs with substantial anti-CMV activity, such as ganciclovir, has dramatically reduced complications associated with post-bone marrow transplant CMV infections. Schmidt et al., New England J. Med. 324:1005–1011, 1991; Goodrich et al., New England J. Med. 325:1601–1607, 1991. Ganciclovir is most effective when administered prophylactically before diagnosis of CMV infection. This approach has several negative consequences in patients, however, including neutropenia and increases in fatal bacterial and fungal diseases. Goodrich et al., Ann. Intern. Med. 118:173–178, 1993. In addition, because of the acute nature of the potential side-effects of ganciclovir treatment, there is a need for increased hospitalization and growth factor administration to treated patients which, coupled with the cost of ganciclovir prophylaxis, increases the total cost of bone marrow transplant after-care. Other treatments available for CMV disease treatment or prophylaxis, such as Foscarnet, Cidofovir or Valganciclovir, also have been used, but also have significant and potentially dangerous side effects that limit their use. Antiviral drug therapy generally can cause significant morbidity and mortality for some patients.
Because human cytomegalovirus is relatively common, yet is associated with some extremely serious health conditions, a considerable effort has been made to study the biology of the virus with the aims of improving diagnosis of the disease as well as developing preventative and therapeutic strategies. Mounting a CD8+ CTL response is believed to be an important mammalian host response to certain acute viral infections. The observations that CMV infection is widespread and persistent, and can become reactivated and clinically evident in the immunosuppressed patient, suggest that virus-specific CTL are involved both in controlling persistent infection and in recovery from CMV disease.
In bone marrow transplant recipients, protection from the development of CMV disease correlates with the recovery of measurable CD8+ CMV-specific class I MHC-restricted T cell responses. Quinnan et al., New Eng. J. Med. 307:7–13, 1982; Reusser et al., Blood 78:1373–1380, 1991. These observations led investigators to carry out clinical trials in which donor-derived CMV-specific CD8+ CTL were infused into bone marrow transplant recipients as an alternative to ganciclovir prophylaxis and therapy. Riddell et al., Science 257:238–241, 1992. The transfer of CD8+ CTL clones to allogeneic bone marrow transplant recipients resulted in detectable CTL-based CMV immunity, and statistically significant diminution of CMV disease. Walter et al., New Eng. J. Med. 333:1038–1044, 1995.
Although successful in application, this approach has the disadvantage that the production of CMV-specific T cells often is problematic. Many culture systems have been developed to generate CMV-specific T cells, such as CMV-infected or retrovirus-infected antigen presenting cells. These antigen presenting cells provide immunodominant CMV antigens, however there is a risk of viral transmission when the cultured T cells are given to severely immunocompromised patients such as bone marrow transplant recipients. Thus, regulatory and safety policies may limit use of virions to stimulate T cells in the clinic.
Other strategies to produce CMV-specific T cells safely include administration of CMV-specific peptide antigens or purified CMV viral protein-pulsed antigen presenting cells, but the high cost and only moderate efficacy of these methods are a concern. To make adoptive immunotherapy treatment widely available to bone marrow transplant patients and other patients, delivery of multiple CMV immunodominant antigens to antigen presenting cells is an important obstacle in the current art to provide expanded CMV-specific T cells in vitro for safe administration to patients. Therefore, a safe, fast and efficient method to prepare CMV-specific T cells for clinical applications have high potential benefits for patients in need of CMV cellular immunity or augmented CMV cellular immunity, such as bone marrow transplant patients.
Another desirable alternative would be to deliver a vaccine derived from CMV that imparts immunity without the need for ex vivo expansion of CMV-specific CTL. No such vaccine presently is available on the market, however. Persons who would benefit from vaccination by compositions according to this invention include, but are not limited to, women of childbearing years, pregnant women, infants, children in a daycare or public school setting, bone marrow transplant recipients and donors, solid organ transplant patients, HIV-positive individuals, coronary angioplasty patients, cancer patients, persons undergoing immunosuppressive therapy or any person at risk for CMV infection or CMV reactivation.
In producing vaccines that are designed to elicit immune response dependent on CD8+ T lymphocytes, it is important to consider that antigens entering the cell through exogenous pathways (pinocytosis, etc.) typically are not processed and presented by Class I MHC molecules. Therefore, methods to introduce proteins directly into the cytoplasm have become one focus of vaccine developers. An approach that has gained favor is to use infection with recombinant vaccinia viruses to deliver and express a large amount of intracellular antigen. The enthusiasm for using vaccinia viruses as vaccines has diminished, however, because these viruses themselves have the potential to cause disease in immunosuppressed people. Another approach to vaccination is to mix antigenic protein with an adjuvant and introduce the mixture under the skin by subcutaneous injection. None of these methods have resulted in production of a clinically useful vaccine to this point. Accordingly, in spite of significant efforts towards identifying the CMV proteins that are recognized by CTL and lead to cellular immunity against the virus, improved methods of preventing and treating CMV infection are needed.
The purpose of (live) viral vaccination is to induce both helper and cytotoxic immunity, which leads to a prolonged and durable memory response against the virus. In the 1970s, Plotkin and co-workers established an attenuated strain of CMV, referred to as Towne, as a proposed therapeutic vaccine. Plotkin et al., J. Infect. Dis. 159:860–865, 1989; Plotkin et al., Ann. Intern. Med. 114:525–531, 1991. Concerns about using live virus, however, have prevented its use generally. In addition, the effectiveness of Towne at preventing CMV transmission has been questioned. Adler et al., J. Infect. Dis. 171:26–32, 1995. Potentially more problematic for an approach using attenuated CMV as a vaccine is the possibility of acquiring infection with a new CMV strain even while having a pre-existing immunity to a different strain. Boppana et al., N. Engl. J. Med. 344:1366–1371, 2001.
Alternative live viral approaches for CMV vaccines have focused on canarypox expressing glycoprotein B (gB) or pp65. Gonczol and Plotkin, Curr. Top. Microbiol. Immunol. 154:255–274, 1990; Adler et al., J. Infect. Dis. 180:843–846, 1999; Berencsi et al., J. Infect. Dis. 183:1171–1179, 2001; Spaete et al., Virology 167:207–225, 1988. These methods have not been successful at eliciting both humoral and cellular immunity to CMV. Studies with poxvirus expressing gB and purified gB also did not reveal any additional benefit. Bernstein et al., J. Infect. Dis. 185:686–690, 2002. The only gB vaccine with any clinical efficacy is a purified gB protein vaccine, however no published information had demonstrated CMV protection. Pass et al., J. Infect. Dis. 180:970–975, 1999. Some DNA vaccine vectors expressing either gB or pp65 have been evaluated in animal models. Endresz et al., Vaccine 19:3972–3980, 2001; Endresz et al., Vaccine 17:50–58, 1999; Pande et al., Scand. J. Infect. Dis. Suppl. 99:117–120, 1995. Thus, although there has been some progress in achieving a clinically useful CMV vaccine, a clear-cut strategy in which both cellular and humoral responses are stimulated from a single vector or other modality has been elusive. Therefore, a comprehensive and multifunctional CMV vaccine or other method is needed in the art, particularly a method to augment CMV immunity in the immunocompromised patient and to prevent reactivation of CMV disease.